Preparation of a Purified Vaccinia Virus Freed from Soluble Antigens

Discussion and summary

The reported experiments form part of a major study of the pattern of virus particle-associated and of soluble antigens, related to vaccinia infection. To study the antigenic composition of the vaccinia virus particle a purified virus material freed from soluble antigen was required. Principally, the method of purification described is a combination of the one used by Marquardt et al(7) and fraction-ation in a sucrose density gradient. Electron microscopy indicated that very little particu-late impurity was present in the virus material banded in the gradient and washed by centrifugation. In the purified material, containing about 2 mg of virus particles per ml, no hemagglutinin and precipitinogenic factors were demonstrable. Ultrasonic treatment immediately preceding the diffusion-in-gel tests was found important as it increased the reactivity of the antigenic preparations.

The lowest concentration of vaccinial pre-cipitinogens necessary for giving a visible reaction in the double diffusion-ingel test is unknown. With a purified LS preparation (11) we found, however, that only 0.6 μg protein was necessary to cause precipitation with a homologous antiserum, indicating that the micro double diffusion test is highly sensitive for demonstration of vaccinial antigen-antibody precipitating systems. The high sensitivity of the micro double diffusion plate test has been documented by Crowle(12).

According to Rondle and Dumbell(13) using a macro plate technique at least 9 precipitating factors are present in cowpox antigenic preparations. The present study demonstrated the existence of at least 7 precipitinogens in crude virus suspensions produced with a rabbit skin adapted strain of vaccinia virus. The presence of additional precipitating factors is indicated by further experiments to be reported.